Cationic lipid: DNA complexes for gene targeting

ABSTRACT

This invention herein describes pharmaceutical compositions and methods for targeted delivery of functional genes into cells and tissues in vivo. The invention discloses DNA:lipid complexes, methods of making such complexes and methods of using such complexes for facilitating the targeted delivery and entry of recombinant expression constructs into cells and tissues in vivo, and particularly delivery of such recombinant expression constructs by intravenous, intraperitoneal or direct injection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A perennial goal in the pharmacological arts has been the development ofmethods and compositions to facilitate the specific delivery oftherapeutic and other agents to the appropriate cells and tissues thatwould benefit from such treatment, and the avoidance of the generalphysiological effects of the inappropriate delivery of such agents toother cells or tissues of the body. Recently, the advent of recombinantDNA technology and genetic engineering has provided the pharmacologicalarts with a wide new spectrum of agents that are functional genescarried in recombinant expression constructs capable of mediatingexpression of these genes in host cells. These developments have carriedthe promise of "molecular medicine", specifically gene therapy, wherebya defective gene could be replaced by an exogenous copy of its cognate,functional gene, thereby alleviating a variety of genetic diseases.

However, the greatest drawback to the achievement of effective genetherapy has been the inability in the art to introduce recombinantexpression constructs encoding functional eukaryotic genes into cellsand tissues in vivo. While it has been recognized in the art as beingdesirable to increase the efficiency and specificity of administrationof gene therapy agents to the cells of the relevant tissues, the goal ofspecific delivery has not been achieved in the prior art.

Liposomes have been used to attempt cell targeting. Rahman et al., 1982,Life Sci. 31: 2061-71 found that liposomes which contained galactolipidas part of the lipid appeared to have a higher affinity for parenchymalcells than liposomes which lacked galactolipid. To date, however,efficient or specific delivery has not been predictably achieved usingdrug-encapsulated liposomes. There remains a need for the development ofa cell- or tissue-targeting delivery system.

Thus there remains in the art a need for methods and reagents forachieving cell and tissue-specific targeting of gene therapy agents,particularly recombinant expression constructs encoding functionalgenes, in vivo.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an improved methods for targeteddelivery of functional eukaryotic genes to cells and tissues in vivo.This delivery system achieves such specific delivery by the formation ofDNA:lipid complexes between nucleic acid comprising a recombinantexpression construct encoding a functional eukaryotic gene or fragmentthereof complexed with a mixture of a cationic lipid and a neutrallipid. Methods of use are also provided. This invention has the specificadvantage of targeted delivery of functional eukaryotic genes into cellsin vivo, achieving effective intracellular delivery of constructsencoding functional genes more efficiently and with more specificitythan conventional delivery systems.

In a first embodiment, the invention provides a pharmaceuticalcomposition, comprising a formulation of a soluble complex of arecombinant expression construct and a mixture of a neutral lipid and acationic lipid in a pharmaceutically acceptable carrier suitable foradministration to an animal by injection. In these embodiments of theinvention, the recombinant expression construct comprises a nucleic acidencoding a protein, the nucleic acid being operatively linked to geneexpression regulatory elements and whereby the protein encoded by thenucleic acid is expressed.

In this first embodiment, the cationic lipid is a nitrogen-containing,imidazolinium-derived cationic lipid having the formula: ##STR1##wherein each of R and R₁ independently is a straight-chain, aliphatichydrocarbyl group of 11 to 29 carbon atoms inclusive. Preferred arethose cations wherein each of R and R₁ independently have from 13 to 23carbon atoms inclusive. In particularly preferred embodiments, thecationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium. Inadditional preferred embodiments, the neutral lipid is cholesterol, andthe 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium andcholesterol are present in the complex at a ratio of 1:1. Furtherpreferred embodiments comprise a recombinant expression constructencoding human CFTR and a mixture of a neutral lipid and a cationiclipid comprises a ratio of DNA to lipid of from about 1:6 to about 1:8(μgDNA:nmoles lipid). Particularly preferred are embodiments where theDNA comprising the recombinant expression construct is present in thecomplex at a concentration of about 0.5 to 1 mg/mL. In further preferredembodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium and theneutral lipid is dioleoylphosphatidylethanolamine, and the1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium anddioleoylphosphatidylethanolamine are present in the complex at a ratioof 1:1. Further preferred embodiments comprise a recombinant expressionconstruct and a mixture of a neutral lipid and a cationic lipidcomprises a ratio of DNA to lipid of about 1:1. Particularly preferredare embodiments where the DNA comprising the recombinant expressionconstruct is present in the complex at a concentration of about 0.5 to 5mg/mL.

In a second embodiment, the invention provides methods for introducing arecombinant expression construct into a cell comprising lung tissue inan animal, the method comprising the step of administering thepharmaceutical composition of claim 1 to the animal by intravenousinjection. In preferred embodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium. Inadditional preferred embodiments, the neutral lipid is cholesterol, andthe 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium andcholesterol are present in the complex at a ratio of 1:1. Furtherpreferred embodiments comprise a recombinant expression construct and amixture of a neutral lipid and a cationic lipid comprises a ratio of DNAto lipid of from about 1:6 to about 1:8. Particularly preferred areembodiments where the DNA comprising the recombinant expressionconstruct is present in the complex at a concentration of about 0.5-1mg/mL.

In another aspect of the second embodiment of the invention is providedmethods for introducing a recombinant expression construct into a cellcomprising spleen tissue in an animal, the method comprising the step ofadministering the pharmaceutical composition of claim 1 to the animal byintravenous injection. In preferred embodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium. Inadditional preferred embodiments, the neutral lipid isdioleoylphosphatidylethanolamine, and the cationic lipid and the neutrallipid are present in a ratio of 1:1. Further preferred embodimentscomprise a recombinant expression construct and a mixture of a neutrallipid and a cationic lipid comprises a ratio of DNA to lipid of about1:1. Particularly preferred are embodiments where the DNA comprising therecombinant expression construct is present in the complex at aconcentration of about 1-2.5 mg/mL.

In further embodiments, the DNA:lipid complex is targeted to peritonealmacrophages by administration by intraperitoneal injection. In theseembodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium, theneutral lipid is cholesterol, the cationic lipid and the neutral lipidare present in a ratio of about 1:1, the complex of a recombinantexpression construct and a mixture of a neutral lipid and a cationiclipid comprises a ratio of DNA to lipid of about 1:1, the DNAconcentration in the DNA:lipid complexes is about 1-2.5 mg/mL. Inadditional embodiments of this aspect of the invention, the DNA:lipidcomplex is targeted to spleen macrophages and administered byintraperitoneal injection. In these embodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium, theneutral lipid is cholesterol, the cationic lipid and the neutral lipidare present in a ratio of about 1:1, the complex of a recombinantexpression construct and a mixture of a neutral lipid and a cationiclipid comprises a ratio of DNA to lipid of about 1:1, the DNAconcentration in the DNA:lipid complexes is about 1 to 2.5 mg/mL.

In this aspect, the invention also provides methods for targeting genetransfer into pancreatic tissue by intraperitoneal injection. Inpreferred embodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium, theneutral lipid is dioleoylphosphatidylethanolamine, the cationic lipidand the neutral lipid are present in a ratio of about 1:1, the complexof a recombinant expression construct and a mixture of a neutral lipidand a cationic lipid comprises a ratio of DNA to lipid of about 1:1, theDNA concentration in the DNA:lipid complexes is about 1.5 to about 2.5mg/mL.

The invention also provides a method of introducing a recombinantexpression construct into a cell comprising a tissue in an animal, themethod comprising the step of administering the pharmaceuticalcomposition of claim 1 to the animal by direct injection. In preferredembodiments, the cationic lipid is1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium and theneutral lipid is cholesterol. Also preferred are mixtures of thecationic lipid and the neutral lipid in a ratio of about 1:1. Preferredcomplexes include a complex of a recombinant expression construct and amixture of a neutral lipid and a cationic lipid comprising a ratio ofDNA to lipid of about 1:1. The preferred DNA concentration in theDNA:lipid complexes is about 1-2.5 mg/mL in this embodiment of theinvention.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the stability of DNA:liposome complexes of theinvention assayed by intravenous administration and lung CAT assays overa period of 11 weeks.

FIG. 2 is a graph of a comparison of chloride efflux in the presence andabsence of stimuli in cells transfected with human CFTR-encoding plasmidvectors complexed with EDMPC:cholesterol.

FIG. 3 is a schematic representation of the plasmid p4119.

FIG. 4 is a histogram showing that mice administered CAT-encodingplasmids complexed with DOTIM cholesterol liposomes exhibited CAT geneexpression in the lung.

FIG. 5 is a histogram showing β-galactosidase expression in mouse lungin 9 experimental mice administered β-galactosidase-encodingDNA:liposome complexes of the invention.

FIG. 6 is a schematic representation of the plasmid pMB19.

FIG. 7 is a graph showing long-term, persistent expression of CATactivity in mouse lung obtained after intravenous administration ofCAT-encoding DNA:liposome complexes of the invention.

FIG. 8 is a histogram showing CAT gene expression in brain afterCAT-encoding DNA:liposome complexes of the invention were administeredintracranially.

FIG. 9 is a histogram comparing tissue specificity of CAT geneexpression in DNA:liposome complexes administered intravenously (samples1 and 2) or intraperitoneally (samples 4 and 5).

FIG. 10 is a histogram showing formulation-dependent variability in theextent of spleen expression of CAT after intravenous administration ofDNA:liposome complexes of the invention.

FIG. 11 is a histogram showing human HLA antigen expression in bonemarrow, spleen and lymph nodes following intravenous administration ofvarious formulations of DNA:liposome complexes of the invention.

FIG. 12 is a representation of tissue-specific targeting of CAT-encodingDNA complexed with different liposome complexes and administeredintravenously.

FIG. 13 is a histogram illustrating CAT gene targeting to peritonealmacrophages after intraperitoneal injection.

FIG. 14 is a histogram showing macrophage-specific targeting byadministration of CAT-encoding DNA using DNA:liposome complexes of theinvention.

FIG. 15 is a histogram showing pancreas-specific targeting byadministration of CAT-encoding DNA using DNA:liposome complexes of theinvention.

FIG. 16 is a histogram showing spleen-specific targeting byadministration of CAT-encoding DNA using DNA:liposome complexes of theinvention.

FIG. 17 is a representation of tissue-specific targeting of CAT-encodingDNA complexed with different liposome complexes and administeredinterperitoneally.

FIG. 18 is a histogram showing CAT gene expression in human prostatetissue in which CAT-encoding DNA using DNA: liposome complexes of theinvention were directly administered ex corpora.

FIG. 19 is a histogram showing a comparison of spleen-specific andlung-specific targeting of DNA:liposome complexes of the invention usingintravenous and intraperitoneal routes of administration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions of matter and methods forfacilitating the entry into cells of nucleic acids, particularlyrecombinant expression constructs encoding functional eukaryotic genes.For the purposes of this invention, the term "recombinant expressionconstruct" is intended to encompass a replicable DNA constructcomprising a nucleic acid encoding a functional gene or fragmentthereof, operably linked to suitable control sequences capable ofeffecting the expression of the gene in a suitable host cell. Expresslyintended to fall within the definition of a "gene" are embodimentscomprising cDNA and genomic DNA embodiments of functional eukaryoticgenes, as well as chimeric hybrids thereof. Also intended to fall withinthe scope of the recombinant expression constructs of the invention arefragments of such genes which, when expressed, may inhibit or suppressthe function of an endogenous gene in a cell, including, inter alia,antisense gene fragments or ribozymes.

In the recombinant expression constructs as provided by the presentinvention, the need for such control sequences will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation. See, Sambrook et al.,1990, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press:New York).

Vectors useful for practicing the present invention include plasmids,viruses (including phage), retroviruses, and integratible DNA fragments(i.e., fragments integratable into the host genome by homologous ornon-homologous recombination). Also useful are vectors which replicateautonomously in host cells. Suitable vectors will contain replicon andcontrol sequences which are derived from species compatible with theintended expression host cell.

The recombinant expression constructs of the present invention areuseful in gene therapy, and specifically, delivering exogenous copies ofa defective gene to a specific tissue target in vivo. See generallyThomas & Capecchi, 1987, Cell 51: 503-512; Bertling, 1987, BioscienceReports 7: 107-112; Smithies et al., 1985, Nature 317:230-234.

The invention provides complexes of recombinant DNA constructs encodingfunctional eukaryotic genes or fragments thereof and also comprising amixture of a cationic lipid and a neutral lipid. For the purposes ofthis invention, the term "cationic lipid" is intended to encompasslipids which are positively charged at physiological pH, and moreparticularly, constitutively positively charged lipids comprising, forexample, a quaternary ammonium salt moiety. Expressly within theteachings of the present invention are co-owned and co-pending U.S.patent applications, Ser. Nos. 08/245,737, filed May 18, 1994;08/248,005, filed May 24, 1994; 08/247,963, filed May 24, 1994;08/157,637, filed Jun. 7, 1994; and International Patent ApplicationNos. PCT/US94/13428, filed Nov. 17, 1994; PCT/US94/13363, filed Nov. 17,1994; and PCT/US94/13362, filed Nov. 17, 1994, which are all hereinincorporated by reference in their entireties.

Specifically, the invention provides nitrogen-containing, imidazoliniumderived cationic lipids having the formula: ##STR2## wherein each of Rand R₁ independently is a straight-chain, aliphatic hydrocarbyl group of11 to 29 carbon atoms inclusive. Preferred are those cations whereineach of R and R₁ independently have from 13 to 23 carbon atomsinclusive. The R and R₁ groups are saturated or are unsaturated havingone or more ethylenically unsaturated linkages and are suitably the sameor are different from each other. Illustrative R₁ groups includelauroyl, myristoyl, palmitoyl, stearoyl, linoleoyl, eicosanoyl,tricosanoyl and nonacosanoyl. In preferred embodiments, the cationiclipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium(abbreviated as DOTIM herein).

The cationic lipids comprising the liposome formulations of theinvention can be synthesized by a rearrangement reaction disclosed inco-owned and co-pending U.S. Ser. No. 08/247,963, filed May 24, 1994,incorporated by reference herein. This reaction comprises synthesis ofDOTIM from N,N-bis(2-hydroxyethyl)ethylenediamine through anamino-protected diacylated intermediate to the desired product. Themethod in general involves synthesis of an imidazolinium ion by heatinga precursor compound of formula ##STR3## in an organic solvent at atemperature above the boiling point of water, wherein each of R and R₁independently represents an organic group such that the precursorcompound is soluble in the solvent and the R and R₁ are stable againstreaction in the solvent at the temperature. The general synthetic method(including some non-essential steps directed to preferred embodimentsand preliminary reactions prior to the key step) can be found in U.S.Ser. No. 08/247,963; this cationic lipid is also commercially available(Avanti Polar Lipids, Ala.).

Cationic lipids are particularly useful as carriers for anioniccompounds, particularly polyanionic macromolecules such as nucleicacids. As cationic lipids are positively charged, a tight charge complexcan be formed between a cationic lipid carrier and a polyanionic nucleicacid, resulting in a lipid carrier-nucleic acid complex which can beused directly for systemic delivery to a mammal or mammalian cell.

Neutral lipids are characterized in contrast to the cationic lipids ofthe invention and are characterized as being electrochemically neutral,although this definition does not preclude protonation of such lipids toproduce a positively-charged salt under certain conditions. Expresslyincluded within this definition are, inter alia, cholesterol anddioleoylphosphatidyl ethanolamine.

Complexes of DNA and mixtures of cationic and neutral lipids of theinvention are characterized by a number of parameters intrinsic to theformation of such complexes. These include the identity of the cationiclipid and the neutral lipid; the ratio of cationic lipid to neutrallipid; concentration of DNA in the complex; the ratio of DNA to lipid;DNA purity; cationic liposome size; methods of preparing liposomes; themethods of preparing the DNA:liposome complexes; and other variables.Preferred combinations of cationic and neutral lipids include1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium andcholesterol and1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium anddioleoylphosphatidyl ethanolamine. A preferred ratio of these lipids is1:1. DNA concentration in the complexes is from about 0.5 mg/mL to about5 mg/mL, more preferably from about 0.5 mg/mL to about 2.5 mg/mL.DNA:lipid ratios are preferably from about 1:1 for formulations to beinjected intraperitoneally, to from about 1:6 to 1:8 for preparations tobe injected intravenously. DNA purity has a direct effect on liposomecomplex formation, but DNAs having a purity of about 15% to about 90%are appropriate for complex formation.

The various lipid carrier-nucleic acid complexes wherein the lipidcarrier is a liposome are prepared using methods well known in the art.Mixing conditions can be optimized by visual examination of theresultant lipid-DNA mixture to establish that no precipitation occurs.To make the lipid-DNA complexes more visible, the complexes that can bestained with a dye which does not itself cause aggregation, but whichwill stain either the DNA or the lipid. For example, Sudan black (whichstains lipid) can be used as an aid to examine the lipid-DNA mixture todetermine if aggregation has occurred. Particle size also can be studiedwith methods known in the art, including electronic microscopy, laserlight scattering, Coulter™ counting/sizing, and the like. Standard-sizebeads can be included as markers for determining the size of anyliposomes or aggregates that form. By "lipid carrier-nucleic acidcomplex" is meant a nucleic acid sequence as described above, generallybound to the surface of a lipid carrier preparation, as discussed below.The lipid carrier preparation can also include other substances orcofactors. Furthermore, the lipid carrier-nucleic acid complex caninclude targeting agents to deliver the complex to particular cell ortissue types. Generally, the nucleic acid material is added to asuspension of preformed liposomes which may be multi-lamellar vesicles(MLVs) or small unilamellar vesicles (SUVs), usually SUVs formed bysonication. The liposomes themselves are prepared from a dried lipidfilm that is resuspended in an appropriate mixing solution such assterile water or an isotonic buffer solution such as 10 mM Tris/NaCl or5% dextrose in sterile water and sonicated to form the liposomes. Thenthe preformed lipid carriers are mixed directly with the DNA.

Mixing and preparing of the lipid-DNA complex can be critically affectedby the sequence in which the lipid and DNA are combined. Generally, itis preferable (to minimize aggregation) to add the lipid to the DNA atratios of DNA:lipid of up to 1:2 inclusive (microgram DNA:nanomolescationic lipid). Where the ratio of DNA:lipid is 1:4 or higher, betterresults are generally obtained by adding the DNA to the lipid. In eithercase, mixing should be rapidly achieved by shaking or vortexing forsmall volumes and by use of rapid mixing systems for large volumes. Thelipid carrier and DNA form a very stable complex due to binding of thenegatively charged DNA to the cationic lipid carriers. SUVs find usewith small nucleic acid fragments as well as with large regions of DNA(≧30 kb).

Aggregation of the lipid carrier-nucleic acid complex is prevented bycontrolling the ratio of DNA to lipid carrier, minimizing the overallconcentration of DNA:lipid carrier complex in solution, usually lessthan 5 mg DNA/mL solution, and avoiding the use of chelating agents suchas EDTA and/or significant amounts of salt, either of which tends topromote macro-aggregation. The preferred excipient is water,dextrose/water or another solution having low or zero ionic strength.Further, the volume should be adjusted to the minimum necessary forinjection into the host mammal, while at the same time taking care notto make the solution too concentrated so that aggregates form.

Liposomes of the invention may be sized in accordance with conventionaltechniques, depending upon the desired size.

The DNA:lipid complexes of the invention have utility in mediating theefficient delivery of the recombinant expression constructs of theinvention, encoding functional eukaryotic genes of fragments thereof,into eukaryotic, preferably mammalian, most preferably human cells.DNA:lipid complexes of the invention are useful for achieving genetransfer in vitro using established techniques. More importantly, theDNA:lipid complexed provided by this invention, and the methods ofadministering the DNA:lipid complexes provided herein, are capable ofspecifically delivering recombinant expression constructs of theinvention to particular tissues and cells comprising those tissues invivo, thereby providing targeting of these genes to specific tissues.These properties of the pharmaceutical compositions and methods of thepresent invention provide for real gene therapy, whereby a particulardeficient gene is restored by the introduction of a functional copy ofthe normal cognate gene into the cells of the affected tissue, withoutdeleterious and unpredictable results from inappropriate introduction ofthe construct into other cells and tissues of the body nonspecifically.

Thus, the invention provides methods and pharmaceutical compositionshaving a number of advantages over the prior art. The liposomes andlipid complexes of the invention have been extensively studied inhumans, and are non-immunogenic, relatively non-toxic, andnon-infectious. These complexes are stable, as illustrated by theexperimental results shown in FIG. 1. A particular DNA:liposome complex(DOTIM:Cholesterol (1:1) complexed with a CAT-encoding plasmid at a DNA:lipid ratio of 1:6 and a DNA concentration of 0.625 mg/mL) was preparedand tested weekly over 11 weeks by injection into the tail vein of ICRmice. CAT activity was then determined in mouse lung using protocolsdescribed in detail below. The Figure shows results demonstrating thatthis preparation was stable over the course of the experiment, wherebysubstantially identical levels of CAT gene expression were obtained atall time points tested.

The DNA:lipid complexes of the invention have additional advantages overthe prior art. Recombinant expression constructs of any practicable sizecan be used, there being no limitation on large plasmid size due to theabsence of packaging the DNA into the genome of a vector organisms likea retrovirus or an adenovirus. Gene transfer can be achieved innon-dividing cells, unlike prior art systems which relied on viralvectors whose life cycle required the infected cells to be dividing. Inaddition, the specific formulation of the DNA:lipid complexes of theinvention can be altered to affect targeting and duration of thegene-expression effect. The DNA:lipid complexes of the invention arealso amenable to many delivery routes, and are less likely to encounterthe types of severe regulatory requirements anticipated for viral-baseddelivery systems.

The DNA:lipid complexes of the invention may be administered to ananimal to effect delivery of functional genes into specific tissues byany appropriate therapeutic routine, including intravenous,intraperitoneal, subcutaneous, or intramuscular injection; directinjection into the target tissue(s). Typically, the DNA:lipid complexesof the invention are injected in solution where the concentration of theDNA to be delivered dictates the amount of the complex to beadministered. This amount will vary with the tissue to be targeted andthe effectiveness of the targeted DNA, the required concentration forthe desired effect, the number of administrations, and the like.

The methods and pharmaceutical compositions of the invention thus areparticularly useful and appropriate for introducing functional humangenes, particularly human CFTR, to lung tissue. These methods andpharmaceutical compositions thus have utility in the treatment of humandiseases, including cystic fibrosis and chronic bronchitis.

The following Examples illustrate certain aspects of the above-describedmethod and advantageous results. The following examples are shown by wayof illustration and not by way of limitation.

EXAMPLE 1 Preparation of DOTIM:Cholesterol (1:1) Small UnilamellarVesicles

To a 1L round bottom flask was added 500 μmoles cholesterol dissolved inan excess of chloroform and then 500 μmoles DOTIM also dissolved in anexcess of chloroform. The amount of DOTIM was determined by phosphorusassay and not simply on the basis of the dry weight of the reagent.

After brief, gently mixing, the flask was attached to a rotaryevaporating apparatus and chloroform withdrawn under slow speed andwater vacuum conditions until almost all of the solvent was evaporated.Evaporation was completed at maximum rotation speed using a vacuum pumpto completely dry the lipid mixture to a thin film on the wall of theround bottom flask.

As an intermediate step to the formation of the title composition,multilamellar vesicles (MLVs) were prepared from this film by theaddition of 16 mL endotoxin-free water to the flask, which was thenwarmed to 37° C. in a water bath with gentle hand-swirling. The MLVsthus formed were removed from the flask using a 9" Pasteur pipette andtransferred to a 20 mm screw cap tube at room temperature. The flask wascleared of any remaining MLVs by washing with an additional 4 mLendotoxin-free water, which was added to the 16 mL previouslytransferred from the flask. These solutions were mixed, and aliquottedequally into 20 16 mL screw cap tubes using a Pasteur pipette.

MLVs were converted into the SUVs of the title composition bysonication. Each of the 16 mL screw cap tubes containing MLVs wereplaced individually into a sonicating water bath maintained at 36° C.for 5 min, and the temperature of the bath checked between theintroduction of each tube. Sonicated droplets within each tube werecollected by brief vortex mixing, and the individual solutions of SUVswere then combined into a single 20 mm screw cap tube using a 9" Pasteurpipette, and then filtered using a 0.2 micron disposable filter(Nalgene). Finally, an amount of an endotoxin-free solution of 25%dextrose in water, equal to one-quarter of the final volume of SUVs, wasadded to the tube of SUVs. This resulted in a suspension of SUVscomprising 20 mM DOTIM and 20 mM cholesterol (40 mM total lipid) in a 5%dextrose solution, which was kept at 4° C. until use.

EXAMPLE 2 Large Scale Plasmid DNA Preparation

Plasmid DNA was prepared in large-scale (i.e., milligram) quantitiesusing a modification of the alkaline lysis procedure (Sambrook et al.,1990, ibid.). Briefly, bacteria comprising a single colony were grownfor 12-18 hours or overnight in 15 mL TB broth (47 g/L TB (SigmaChemical Co., St. Louis, Mo.)/ 8% glycerol) supplemented with 100 μg/mLcarbenicillin at 37° C. with shaking (250 rpm). 2-2.5 mL of this culturewas then added to 40 mL TB (supplemented with 100 μg/mL carbenicillin)in each of six 2L flasks (for a total of 2.4L culture) and grown at 37°C. with shaking overnight (16-18 h).

After overnight growth, bacteria were collected by centrifugation for 10min. at 4° C. in a Beckman J2-MI centrifuge equipped with a JA-10 rotor.The bacterial pellet in each centrifuge bottle was gently resuspended in20 mL of an ice-cold solution of 50 mM dextrose in 25 mM HCl buffer(pH8)/10 mM EDTA. To the resuspended bacterial cell pellets were added40 mL of a freshly-made solution of 0.2N NaOH/1% sodium dodecyl sulfateat room temperature, resulting in cell lysis upon gentle agitation ofthis mixture on ice for about 5 min. After the added lysis solution hasbeen thoroughly mixed into the bacterial suspension and the cells lysed,the mixture was allowed to stand at room temperature for 5 min. To thismixture of lysed bacteria was added 20 mL of an ice-cold solution of 3Mpotassium acetate, which was mixed into the lysed bacterial solutiongently by hand and then stored on ice for 10 min. A flocculent whiteprecipitate formed, comprising bacterial chromosomal DNA, RNA andSDS/protein/membrane complexes, which were cleared from the solution bycentrifugation at 8000 rpm for 15 min at 4° C. in the JA-10 rotor asabove.

After centrifugation, the supernatant was transferred with filteringthrough Miracloth to 250 mL centrifuge bottles, and 50 mL isopropanoladded at room temperature, mixed and incubated for 10 min. The plasmidDNA precipitate was recovered by centrifugation at 5000 rpm for 10 minat room temperature in a JA-14 rotor (Beckman). The alcohol-containingsupernatant was decanted and residual supernatant removed by vacuumaspiration.

The plasmid DNA pellets were resuspended in 6 mL of a solution of 6 mMTris-HCl (pH8) and transferred to 50 mL centrifuge tubes upondissolution. To each tube was added and equal volume of cold (-20° C.)5M LiCl, the solutions mixed by hand and then centrifuged at 8000 rpmfor 10 min at room temperature in a JA-20 rotor (Beckman). Thesupernatant solution from each tube was transferred to a fresh tube andthe plasmid DNA then re-precipitated by the addition of an equal volumeof isopropanol, mixed and collected by centrifugation at 5000 rpm for 10min at room temperature in a JA-20 rotor. The alcohol-containingsupernatant solution was then decanted, residual alcohol removed byaspiration, and the plasmid DNA pellets allowed to air dry for 5 min.

Contaminating bacterial RNA was removed from the plasmid DNA bydissolving the pellets in 1 mL 10 mM Tris-HCl (pH8), adding about0.5-0.75 kg of pancreatic RNase per mL, followed by incubating themixture at 37° C. for 1 h. Disappearance of RNA was determined byethidium bromide-stained agarose gel analysis (see Sambrook et al.,ibid.).

Plasmid DNA was purified by phenol-chloroform extraction. Briefly, toeach aliquot of plasmid DNA solution was added an equal volume ofTris-saturated phenol:chloroform (1:1), the immiscible solutions mixedby vortexing, and centrifuged in a laboratory tabletop microfuge for 5min at room temperature. The aqueous (upper) layer was removed,transferred to a fresh microfuge tube, and extraction withphenol:chloroform repeated at least twice. These extractions werefollowed by two extractions of the aqueous layer with Tris-saturatedchloroform. Plasmid DNA was concentrated by precipitation, with theaddition of 5M sodium acetate to a final concentration of 0.3M and theaddition of two volumes of cold (-20° C.) absolute ethanol. DNA wasallowed to precipitate in this solution at -20° C. for 1 h or overnight.

After precipitation, plasmid DNA was collected by centrifugation atabout 6000 rpm in a clinical microcentrifuge. The alcohol-containingsupernatant was aspirated by vacuum, and the pellet washed twice with70% ethanol/water (4° C.). The washed pellets were air dried for atleast 30 min. Plasmid DNA pellets were dissolved in a total of 6 mL of asolution of 10 mM Tris-HCl (pH8), and concentration determined byspectrophotometric analysis of a 1-to-200 dilution of the recoveredplasmid at A₂₆₀.

EXAMPLE 3 Preparation of DNA:Liposome Complexes

DOTIM:Cholesterol:Plasmid DNA liposomes were prepared as follows. ADOTIM:Cholesterol mixture (1:1, 20 μmoles/μL each lipid) was prepared asdescribed in Example 1 above. Complexes with plasmid DNA were preparedin DNA:liposome ratios of 1:1 and 1:6. DNA and DOTIM:Cholesterol wereeach first brought from storage conditions (-20° C. for DNA, 4° C. forliposome formulations) to room temperature before use over the course ofabout 1.5 h. DNA concentration in the liposome preparations wereoptimally 100-550 μg/200 μL complex (for ratios of 1:1 DNA:liposomes)and 100-150 μg/μL complex (for ratios of 1:6 DNA:liposomes). DNAconcentrations were typically determined just prior to DNA:liposomecomplex formation, by ultraviolet spectrophotometry as described inExample 2. DOTIM:Cholesterol mixtures were typically used at a totallipid concentration of 40 μmole/mL, corresponding to 20 μmole/mL DOTIMand 20 μmole/mL cholesterol.

DNA:liposome complexes were prepared from these reagents as follows.Each component was prepared in individual microfuge tubes to a totalvolume per tube of 100 μL. An appropriate amount of DNA (equivalent to afinal DNA concentration of 500 μg DNA/mL complex) was added to one tube,and brought to volume with water or a solution of 5% dextrose in water.The appropriate amount of the DOTIM:Cholesterol mixture (100 nmoleslipid/100 μg DNA at a 1:1 ratio; 600 nmoles lipid/100 μg DNA at a 1:6ratio) was added to a second tube, and water or a solution of 5%dextrose in water was added to bring this solution to a total volume of100 μL. The contents of the lipid-containing tube were mixed byvortexing for about 2 sec, while the contents of the DNA-containing tubewere mixed gently using a 1 mL pipettor. The contents of the lipidmixture-containing tube were then added to the DNA-containing tube usinga 1 mL pipettor. It was found that it was essential that this additionwas performed slowly, in a constant stream, to the top of the DNAsolution in tube A. As the lipid solution mixed with the DNA, formationof the DNA:liposome complex was detected by the solution becomingslightly cloudy and opalescent. It was also determined that, at thisstage, the mixture could not be vigorously mixed (for example, byvortexing) without seriously compromising the integrity and usefulnessof the complexes so formed; however, it was advantageous to gently mixthe entire contents of the tube 3-4 times after completion of additionof the lipid mixture to the DNA mixture.

After the complexes were formed, the final concentration of DNA wasdetermined by ultraviolet spectrophotometry as described above, and thesize of the DNA:liposome complexes determined by light scatteringmeasured at 400 nm.

EXAMPLE 4 Preparation of Tissue Samples for CAT Assay and ProteinDetermination

Tissues were prepared for assay as follows. Experimental animals wereeuthanized quickly and humanely. Mice were typically placed in a killbox flooded with CO₂ for 2-3 min. Tissues were harvested by dissectionand weighed, and then placed in 1 mL cold homogenation buffer (250 mMTris/5 mM EDTA) supplemented with PMSF (35 μg/mL) andLeupeptin/Aprotinin (5 μg/mL). Tissues were then homogenized for 20-30sec using a tissue disruptor (such as a Polytron) until a uniformhomogenate was obtained. This homogenate was then transferred to acentrifuge tube and quickly frozen on dry ice (or at -70° C.) and thenthawed at 37° C. Insoluble debris was cleared from the homogenate bycentrifugation at 10,000 g for 5-10 min. 50 μL of the resultingsupernatant solution was aliquotted into a microfuge tube and stored at-70° C. until used for protein determinations.

The remainder of the supernatant was heat inactivated at 65° C. for 15min and recentrifuged at 10,000 g for 10 min, and stored at -70° C.until use for CAT assay determinations.

To perform CAT assays, samples were analyzed in parallel with a seriesof standard CAT activity samples. From the standards was developed astandard curve of CAT activity versus CAT protein, which was used todetermine the level of CAT protein expression in tissue samples based onthe observed CAT activity in tissue homogenates. To prepare the standardcurve, serial dilutions of CAT enzyme were prepared ranging from 0.1 to0.000025U. These standards were prepared in a reaction mixtureconsisting of 50 μL BSA buffer (250 mM Tris/5 mM EDTA/2 mg/mL BSA,Fraction V (U.S. Biochemical, Cleveland, Ohio), 5 μL standard CAT enzyme(and appropriate dilutions; obtained from Sigma Chemical Co, St. Louis,Mo.), 50 μL ¹⁴ C-labeled chloramphenicol (New England Nuclear; diluted1:10 in BSA buffer prior to use) and 25 μL n-butyryl-CoA (Sigma). Tissuesamples were prepared identically, with the exception that 30 μL oftissue homogenate was substituted for the 5 μL of standard CAT enzymeactivity. Samples were incubated at 37° C. for 2 h. After thisincubation, 300 μL of mixed xylenes (Aldrich Chemical Co.) were added toeach tube, vortexed for 30 sec, and centrifuged for 3 min at 10,000 rpmin an IEC centrifuge equipped with a 24-slot rotor. The mixed xylene(upper) phase of each sample tube was transferred to a fresh microfugetube and 750 μL homogenation buffer added. The samples were thenvortexed and centrifuged as described above.

200 μL of the upper phase from each tube were transferred to liquidscintillation vials and 0.5 mL scintillation cocktail (Ready-Safe,Beckman) added. The amount of CAT-specific radioactivity in each samplewas determined by liquid scintillation counting assay.

EXAMPLE 5 X-Gal Staining of Tissue Samples

Tissue samples were stained with X-gal(5-bromo-4-chloro-3-indolyl-α-_(D) -galactopyranoside using thefollowing protocol. Tissues are fixed by immersion for 0.5-1 h on ice infreshly-made fixative solution (2% neutral buffered formalin/0.02%gluteraldehyde/0.02% Nonidet-P40). After fixation, tissues were rinsedtwice at 4° C. in a solution of 2 mM MgCl₂ /0.1% desoxycholate/0.2%NP-40 in 10 mM phosphate-buffered saline (PBS; pH 7.3). Tissues werethen stained using rinse solution supplemented with 1 mg/mL X-Gal (U.S.Biochemical), 5 mM ferricyanide and 5 mM ferrocyanide. Tissues werestained for 12-48 h at 37° C. or room temperature. After staining,tissues are rinsed in PBS. Tissues were then frozen and sectioned orfixed in 70% ethanol, embedded in paraffin and sectioned.

Protein determinations were performed using a dye binding assay (BCAPRotein Assay Reagent, Pierce Chemical Co.). The Pierce reagent wasprepared by mixing 50 parts of Reagent A with 1 part Reagent B asprovided by the supplier. 100 μL of this prepared reagent werealiquotted into each well of a 96 well microtiter plate. 100 μL of asolution containing 20 μg BSA were added to the first well of the firstrow (i.e., well A1) and 100 μL of a 1:2 to 1:8 dilution of each tissueextract were added to the other wells in the row. Serial dilutions atratios of 1:2 were made in each of the adjoining rows consecutivelyusing the wells in the preceding row. Typically, 96-well plates having12 wells/row resulted in 6 serial dilutions (1 to 1/64); the last row isa blank loaded with PBS as a control. The plates were incubated at 37°C. for 30 minutes, and the extent of dye binding determinedspectrophotometrically as absorbance at 562 nm. Protein concentrationsin sample wells were determined in comparison with a standard curvegenerated using the OD readings from the serial dilutions of the BSAstandard.

EXAMPLE 6 Microplate Assay for β-galactosidase Expression in Tissues

Tissue was homogenized in an appropriate volume of homogenization buffer(250 mM Tris/5 mM EDTA) (e.g., 300 μL were used to homogenize a mouselung). The homogenate was then incubated on ice for 30 min andcentrifuged in a microcentrifuge for 10 min at 13,000 rpm to clear thehomogenate of insoluble debris. Supernatants from these homogenates werecollected and assayed as follows.

Microplates were prepared for these analyses as follows. For each plateto be covered, 50 μL of anti-β-galactosidase monoclonal antibody wasdiluted in 5 mL of 5 mM sodium bicarbonate buffer (pH 9.4). 50 μL of thediluted antibody solution was added to each well of a microtiter plate(e.g., Immulon 3, Dynatech), the plate sealed and incubated overnight at4° C. After overnight incubation, 200 μL BLOTTO solution (5% v/v nonfatdry milk and 0.2% Tween-20 in PBS) were added to each well and incubatedfor 1 h at room temperature. The BLOTTO solution was then removed andthe plates washed three times with a solution of PBS/0.2% Tween-20, withthe exception that the first row was not washed with this solution. 100μL of a standard solution of 10 mU/mL β-galactosidase were added to thefirst well of the first row (i.e., well A1) and 100 μL of each tissueextract were added to the other wells in the row. Serial dilutions atratios of 1:2 were made in each of the adjoining rows consecutivelyusing the wells in the preceding row. Typically, 96-well plates having12 wells/row resulted in 6 serial dilutions (1 to 1/64); the last row isa blank loaded with PBS as a control. The plates were incubated at roomtemperature for 1 h, and then washed three times with a solution ofPBS/0.2% Tween-20 as above.

To develop the assay, 100 μL of CPRG assay buffer (2.5 mg/mL chlorphenolred-β-_(D) -galactopyranoside monosodium salt (CPRG)/ 1.8 mg/mL MgCl₂/7.1 μL/mL 2-mercaptoethanol in PBS) were loaded into each washed welland the plates then incubated at 37° C. for 2 h. The extent ofβ-galactosidase expression was then determined spectrophotometrically asabsorbance at 562 nm.

Whole tissues and tissue sections were assayed using a modification ofthis protocol. Frozen tissue or tissue sections were fixed by immersingthe frozen tissues in fixative solution (2% neutral bufferedformalin/0.02% glutaraldehyde/0.02% NP-40) without thawing. Tissues wereincubated in fixative solution for 2 h at room temperature with gentleagitation. After incubation, the tissues were rinsed twice with PBS,then incubated at 37° C. overnight in X-Gal staining solution (5 mMpotassium ferricyanide/5 mM potassium ferrocyanide/0.01% sodiumdesoxycholate/0.02% NP-40/1 mg/mL X-Gal in PBS, supplemented with MgCl₂to 20 μM immediately before use). After staining, tissues were washedtwice with PBS, and then embedded in paraffin or quick frozen forsectioning and histochemical analysis.

EXAMPLE 8 Detection of Functional CFTR Expression in Transfected CellsUsing a Chloride Efflux Assay

A chloride ion efflux assay was used to detect functional expression ofCFTR in transfected cells.

About 24 h prior to introducing CFTR into cells, cells were split into a6-well tissue culture dish, each well receiving 1 mL of 10 mL of thecells on the dish and 3 mL media. Cells were returned to the incubatorand allowed to grow overnight at 37° C./5% CO₂, or until they were about70-80% confluent. For assay, media were removed from the wells and eachwell was washed with 2 mL serum-free media. 1 mL of serum-free media wasthen added per well, and the cells incubated at 37° C. for 1-2 h. 200 μlof a DNA-lipid complex comprising a recombinant expression constructencoding CFTR were then added to each well and incubated at 37° C. for6-8 h. After this incubation, media were removed from each well, thewells were washed twice with 2 mL serum-free media and incubated in 4 mLserum-containing media at 37° C. for 48 h.

The chloride ion efflux assay was performed as follows. Media wereaspirated from each of the wells containing cells treated with DNA-lipidcomplexes, and washed twice with efflux solution (135 mM NaCl/2.4 mM K₂HPO₄ /0.6 mM KH₂ PO₄ /1.2 mM CaCl₂ /1.2 mM MgCl₂ /10 mM glucose/10 mMHEPES (pH 7.4)). Cells were then incubated with 1 mL efflux solutioncontaining Na³⁶ Cl at a final concentration of 2.5 μCi/mL ³⁶ Cl⁻ for 2 hat 37° C. After incubation, the ³⁶ Cl⁻ -containing efflux solution wasaspirated from the cells and the cells then washed each of 4 times with1 mL efflux solution. The cells were then incubated with 1 mL effluxsolution for 3 min at room temperature, and the efflux solution thenremoved from the cells and transferred into a scintillation vialcontaining 5 mL scintillation cocktail. A fresh aliquot of effluxsolution was added to each well and incubated for an additional 3 min.After each incubation, efflux solution was transferred to ascintillation vial containing 5 mL scintillation cocktail, and a fresh 1mL aliquot of efflux media was added to the cells and incubated for 3min. These steps of the assay were repeated ten times for a total of 30min. In certain of the wells, ³⁶ Cl⁻ ion efflux was stimulated byincubating these cells in the presence of 40 μM Forskolin (Sigma), 500μM cpt-cAMP (Sigma), and 100 μM IBMX (Sigma) in efflux solution, effluxbeing stimulated at repetitions 3 through 7.

The extent of ³⁶ Cl⁻ ion efflux over this period was determined byscintillation counting, and the basal rate of ³⁶ Cl⁻ ion efflux comparedwith the rate of efflux in cells stimulated by Forskolin/cpt-cAMP/IBMX.Extent of efflux was normalized relative to the amount of ³⁶ Cl⁻ ionremaining inside the cells after the 30 min incubation. This quantitywas determined by lysing the cells by incubating them with 1 mL ofscintillation fluid for 15 min. The lysate from each well was thentransferred into a scintillation vial, the well washed with 1 mL ofefflux solution which was added to the cell lysate, and the ³⁶ Cl⁻ion-associated radioactivity counted.

The results of one such assay are shown in FIG. 2. Two plasmids encodingCFTR and differing in the details of the construct (see Table I) weretested with (closed circles and boxes) and without (open circles andboxes) stimulation. As is shown in the Figure, stimulation results inthe rapid induction of chloride ion efflux over the basal rate ofefflux, which efflux persists even after the stimulus is removed (timepoints 24-30). These results demonstrate the utility of this assay todetect functional expression of CFTR in heterologous cells, and thusforms an in vitro standard for determining the vigor of differentrecombinant expression constructs in expressing human CFTR.

EXAMPLE 9 Reverse Transcriptase-Polymerase Chain Reaction Analysis

Human CFTR gene expression was assayed using a reverse transcriptasepolymerase chain reaction assay (RT-PCR) on transfected tissue culturecells and whole tissues. These assays were performed using vectorspecific primers and CFTR specific primers. The vector

                  TABLE I                                                         ______________________________________                                        Vectors with the CFTR cDNA                                                    enhancer    promoter  intron polyA    antibiotic                              ______________________________________                                        MB19:  HCMV     HCMV      ppi  ppi      amp                                   MB31:  HCMV     HCMV      ppi  SV40     amp                                   MB65:  HCMV     HCMV      ppi  Nmyc ppi amp                                   MB66:  HCMV     HCMV      ppi  Cmyc SV40                                                                              amp                                   MB76:  HCMV     HCMV      ppi  3xSV40   amp                                   MB77:  --       CC10      ppi  3xSV40   amp                                   MB78:  HCMV     CC10      ppi  3xSV40   amp                                   MB81:  --       CFTR      ppi  3xSV40   amp                                   MB87:  HCMV     CFTR      ppi  3xSV40   amp                                   MB90:  HCMV     HCMV      --   3XSV40   amp                                   MB93:  HCMV     HCMV      pg13 SV40     amp                                   MB97:  HCMV     HCMV      pg13 SV40     amp/tet                               MB113: HCMV     HCMV      pg13 SV40     tet                                   ______________________________________                                    

specific primers used were:

    __________________________________________________________________________    5' AGA TCG CCT GGA GAC GCC AT 3'                                                                   forward primer                                                                (3651-3671 bp in pMB19)                                  and                                                                           5' GCT CCT AAT GCC AAA GGA AT 3'                                                                   reverse primer                                                                (1246-1266 bp in pMB19,                                                       upstream from hCFTR ATG site).                           The CFTR specific primers were used:                                          5' CCT GTC TCC TGG ACA GAA A 3'                                                                    forward primer                                                                (3337-3355 bp in pMB19)                                  and                                                                           5' GTC TTT CGG TGA ATG TTC TGA C 3'                                                                reverse primer                                                                (3651-3671 bp in pMB19).                                 __________________________________________________________________________

Tissues were frozen on dry ice for RT-PCT and stored at -70° C. Tissuesamples were homogenized and used directly in this evaluation.

Briefly, RT-PCR was performed by preparing first-strand cDNA fromcellular RNA isolated from frozen tissues using standard techniques (seeSambrook et al., ibid.), including specifically the use of randomhexamer for priming and MMLV-derived reverse transcriptase. cDNA wasused in PCR reactions performed as follows. The entire 25 μL of thefirst-strand cDNA reaction was mixed with the components of the PCRreaction (under standard conditions; see Innis et al., 1990, PCRProtocols: A Guide to Methods and Applications, Academic Press, NewYork), including 25 μM apiece of each of the specific pairs of PCRprimers. PCR reactions were overlayed with light mineral oil to preventcondensation and then subjected to the following PCR cycling protocol:

    ______________________________________                                         1 cycle     10 min 94° C.                                             30 cycles    1 min 94° C.                                                           2 min 55° C.                                                           3 min 72° C.                                               1 cycle     10 min 72° C.                                                          2 min 27° C.                                              ______________________________________                                    

After completion of the reaction, the apparatus was programmed to takeand hold the reaction mixtures at 4° C.

PCR products were analyzed by electrophoreses in agarose or acrylamidegels. In these assays, the vector-specific primers were expected toyield a band representative of plasmid DNA (485 bp) and a hCFTRRNA-specific band (142 bp). The CFTR-specific primers were expected toyield a DNA fragment band of 334 bp.

EXAMPLE 10 Functional Delivery of CAT Gene Constructs to Cells In Vivo

Functional delivery of a variety of CAT reporter gene constructs wasachieved using different embodiments of the DNA:lipid complexes of theinvention.

A. DOTIM:Cholesterol Formulation I

DOTIM:cholesterol liposomes were prepared as described above in 1:1ratio and used to prepare DNA:lipid complexes. DOTIM:cholesterol (1:1)liposomes were used to make DNA complexes using the chloramphenicolacetyl transferase (CAT) expression vector 4119 (FIG. 3). DNA:lipidcomplexes were prepared having a DNA:lipid ratio was 1:6, and using 125μg of DNA per 200 μL complex. Liposome size was determined by opticaldensity (OD) at 400 nm. A total of 200 μL of the complex were injectedinto the tail veins of 3 ICR mice. At 24 hrs post-injection, tissueswere harvested and prepared for CAT assays as described in Example 4above. Tissues harvested included lung, liver, kidney, spleen, ovary,brain, smooth muscle, heart and ear.

Results of these CAT assays are shown in Tables II and III below. TableII shows CAT activity as total ¹⁴ C-labeled chloramphenicol countsconverted to acetyl and diacetyl forms by CAT expression vector-encodedenzyme activity in lung for each of the three experimental animalstested.

                  TABLE II                                                        ______________________________________                                        animal number  CAT (cpm)                                                      ______________________________________                                        20.1-1         800,000                                                        20.1-2         1,400,000                                                      20.1-3         400,000                                                        ______________________________________                                    

Table III shows CAT assay data from a variety of tissues from one of theexperimental animals (animal 20.1-2). These results demonstrate thatintravenous inoculation of mice in the tail vein withDOTIM:cholesterol:DNA complexes in this formulation results inpreferential targeting of the DNA:lipid complexes to the lungs, with CATactivity in lung tissue representing over 80% of the CAT activitydetected in all mouse tissues tested.

                  TABLE III                                                       ______________________________________                                               tissue                                                                             CAT (cpm)                                                         ______________________________________                                               li   20,000                                                                   sp   63,000                                                                   ki   15,000                                                                   ov    3,000                                                                   br    7,000                                                                   sm   58,000                                                                   he   115,000                                                                  ear    1500                                                            ______________________________________                                         Key:                                                                          liver (li),                                                                   kidney (ki),                                                                  spleen (sp),                                                                  ovary (ov),                                                                   brain (br),                                                                   smooth muscle (sm),                                                           heart (he).                                                              

The results of these experiments are also shown graphically in FIG. 4,which summarizes the results obtained with over 700 experimental andcontrol mice. As can be seen in the Figure, treated mice reproduciblyshowed greater than 1000-fold higher CAT activity in lung of micetreated with the DNA:lipid complexes of the invention comprisingCAT-encoding recombinant expression constructs (a total of 555 mice),compared with control (untreated) mice (a total of 163 mice).

The delivery and uptake into cells of various mouse tissues of the CATplasmid DNA administered as DNA:lipid complexes of the invention byinjection into the tail vein of mice was analyzed by Southern blotanalysis using routine procedures (see Sambrook et al. ibid.). DNA frommouse tissues was extracted and purified and digested with BammIrestriction endonucleases. The resulting DNA restriction fragments wereseparated by agarose gel electrophoresis and transferred to a membraneby capillary action. Such membranes were dried, prehybridized and thenhybridized with a radioactively-labeled, CAT DNA-specific probe (about108-109 dpm/μg) at an appropriate stringency (2X-6X SSC at 62° C.)overnight, washed to high stringency (0.1-0.5X SSC at 65° C.) andexposed to autoradiographic film at -70° C. using intensifying screens.

Results of these experiments are shown in FIG. 5. The lower panel isidentical to the upper panel, but has been allowed to expose the X-rayfilm for a longer period of time. These results of these experimentsdemonstrate that CAT DNA is introduced specifically into lung, withsignificant amounts of DNA uptake in spleen. Much lower amounts of CATDNA were observed in certain other tissues (liver, kidney) but manytissues showed essentially no CAT-specific hybridization, even at thelonger exposure time.

A second series of experiments were performed using this lipidformulation. In these experiments, the DNA construct used was theβ-galactosidase expression vector MB10 (see Table I) that encodes a formof β-galactosidase that is translocated into the nucleus in in vitrostudies. Complexes were formed as described above, and mice wereinjected with 200 μL complexes in the tail vein. The resultingβ-galactosidase levels present in lungs are shown in FIG. 5, whichrepresents the results of experiments with 9 experimental and 1 control(administered liposome only) mouse.

In a third series of experiments, expression of the human CFTR gene wasshown following IV delivery of DNA/DOTIM:Cholesterol complexes. Arecombinant expression plasmid encoding the human CFTR gene (MB19; seeTable I and FIG. 6) was used to make DNA:lipid complexes as describedabove (DNA/lipid ratio of 1:6, 125 μg DNA /200 μL complex). Thesecomplexes were tested by transfection/chloride ion efflux assay in human293 cells in vitro, as described in Example 8, and 200 μL was injectedinto each of ICR 3 mice. Cells and lungs were harvested at 24 hrs. RNAwas made using conventional methods as embodied in kits from eitherStratagene (for cell culture results) or 5'-3' Prime (for lung tissues).Samples were analyzed by RT-PCR as described above in Example 9. In thisanalysis, amplification of plasmid sequences yielded a 484 bp PCRproduct, while amplification of cDNA corresponding to spliced CFTR mRNAfor CFTR yielded a 142 bp PCR product. Similar results were obtainedfrom lungs following IV administration of the CFTR/lipid complexes.

The time course of expression of exogenously added CAT-encoding plasmidin mouse lung was determined. A number of mice were injectedintravenously in the tail vein with DNA/lipid complexes comprising 4119CAT DNA at a 1:6 ratio with DOTIM:Cholesterol at a concentration of 125μg/200 μl. Mice were sacrificed in duplicate over a period of 55 days,and lung tissue analyzed by CAT assay as described above. These resultsare shown in FIG. 7, which indicated that high-level, persistentexpression of the reporter gene construct had been achieved.

Complexes of this DOTIM:DNA formulation were also administered by directintracranial delivery. Complexes were made using CAT expression plasmid4119 and complexed with DOTIM:Cholesterol (1:1) at a ratio of 1:1DNA:lipid at a DNA concentration of 500 μg/200 μL. 200 μL of thesecomplexes were directly implanted intracranially, and the extent of CATactivity is brain tissue analyzed 24 h later. The results of theseexperiments are shown FIG. 8.

The results of these different assays indicated that thisDOTIM:Cholesterol formulation was capable of delivering a variety ofrecombinant expression constructs to the lung after intravenousadministration, as well as by direct injection into a tissue of interest(brain).

B. DOTIM:Cholesterol Formulation II

DOTIM:cholesterol liposomes were prepared as described above in 1:1ratio and used to prepare DNA:lipid complexes. DOTIM:cholesterol (1:1)liposomes were used to make DNA complexes using the chloramphenicolacetyl transferase (CAT) expression vector 4119. DNA:lipid complexeswere prepared having a DNA:lipid ratio was 1:1, and using 200-550 μg ofDNA per 200 μL complex. Liposomes were injected into the tail vein ofICR mice, as described above.

CAT gene expression in lung tissue from mice injected withDOTIM:Cholesterol:DNA complexes prepared at a DNA/lipid ratio of 1:1 wasdetermined. Plasmid 4119 DNA was complexed with DOTIM:Cholesterolformulation of the invention, the complexes having a DNA/lipid ratio of1:1. Tail vein injections were performed and tissues harvested at 24 hrsas described.

The results of these assays are shown in Table IV below.

                  TABLE IV                                                        ______________________________________                                        Amount of DNA/complex                                                                          OD.sub.400 *                                                                          lung expression**                                    ______________________________________                                        200 μg/200 μl                                                                            0.24     39,000                                              300 μg/200 μl                                                                            0.32     9,000                                               400 μg/200 μl                                                                            0.53    500,000                                              500 μg/200 μl                                                                            0.66    700,000                                              550 μg/200 μl                                                                            0.82    1,000,000                                            negative control 0.03        0                                                ______________________________________                                         *light scattering as an estimate of liposome size                             **in cpm of acetylated and diacetylated .sup.14 Clabeled chloramphenicol 

DNA/DOTIM liposomes were made using the plasmid 4119 and DOTIM liposomesat ratios of 1:6 and 1:8, were held at 40° C. for 11 days prior totesting and then tested again at 18 days. The results of CAT expressionassays using these formulations are shown in Table V.

                  TABLE V                                                         ______________________________________                                        DNA/DOTIM ratio time stored                                                                             CAT/lung**                                          ______________________________________                                        1:6             11 days     515,000                                           1:8             11 days   1,050,000                                           1:6             18 days   11,000,000                                          1:8             18 days   3,450,000                                           ______________________________________                                         **in cpm of acetylated and diacetylated .sup.14 Clabeled chloramphenicol 

DOTIM:cholesterol complexes with DNA were also administered byintraperitoneal injection. DNA:liposome complexes administeredintravenously and intraperitoneally were compared, using CAT expressionplasmid 4119 DNA complexed with DOTIM:Cholesterol formulation of theinvention. In these assays, the complexes had a DNA/lipid ratio of 1:1and a DNA concentration of 300-500 μg/200 μL. A total of 1 mL of thesecomplexes was injected intraperitoneally in two mice (mice 4 and 5), 200μL were administered intravenously (mice 1 and 2), and 1 mouse wasadministered a formulation comprising only liposomes. Tissues wereharvested at 48 h post-injection. CAT assays were performed as describedabove in Example 3, and the results of these assays are shown in FIG. 9.

The effect on the efficiency of DNA delivery to tissues in vivo ofintravenously administering different formulations comprising the samemixture of cationic and neutral lipids was determined by comparing theextent of transferred CAT activity observed using the differentformulations. CAT plasmid DNA/DOTIM:DOPE (1:1) complexes were preparedin the following formulations:

A. DNA:Lipid ratio of 1:6 DNA concentration of 0.625 mg/mL

B. DNA:Lipid ratio of 1:1 DNA concentration of 2.5 mg/mL

Each formulation was prepared as described in Examples 1 and 3 above,and were administered by intravenous injection into the tail vein ofcohorts of 3 ICR mice per tested formulation. Liposomes that were notcomplexed with DNA were injected into a separate cohort of 3 mice as acontrol.

Animals were sacrificed 1-2 days after injection and analyzed by CATassay of spleen, heart and lung tissue. The results of these experimentsare shown in FIG. 10. This Figure demonstrates that Formulation Bprovides a consistently higher level of CAT activity in spleen, heartand lung than Formulation A, although it appears that the relativeefficiency of plasmid delivery is about the same for both formulations.

C. Comparison of HLA Gene Delivery using Different DNA:Lipid Complexes

Three different lipid formulations were used to deliver a humanHLA-encoding construct to bone marrow, spleen and lymph node. The threeformulations used were:

A. DOTIM:Cholesterol (1:1) DNA:Lipid ratio of 1:6 DNA concentration of0.625 mg/mL

B. DOTIM:Cholesterol (1:1) DNA:Lipid ratio of 1:1 DNA concentration of 2mg/mL

C. DOTIM:DOPE (1:1) DNA:Lipid ratio of 1:1 DNA concentration of 2 mg/mL

(DOPE is dioleoylphosphatidylethanolamine). Each formulation wasprepared as described in Examples 1 and 3 above, and were administeredby intravenous injection into the tail vein of cohorts of 3 ICR mice pertested formulation. Liposomes that were not complexed with DNA wereinjected into a separate cohort of 3 mice as a control.

Animals were sacrificed 1-2 days after injection and analyzed byhistochemical staining for human HLA expression. Tissues were analyzedfor percentage of cells in the tissue positive for human HLA expressionin the histochemical staining assay. Results of these experiments areshown in FIG. 11, wherein Formulation A is MB102, Formulation B is MB107and Formulation C is MB163. For each formulation tested, some cells ineach tissues were found to stain positive for human HLA expression.Lymph node staining varies most among different administeredformulations, with DOTIM:Cholesterol at the higher (2 mg/mL) DNAconcentration providing the most human HLA positive cells, and theDOTIM:DOPE formulation providing the least human HLA positive cells. Theresults in spleen were less variable, with the DOTIM:Cholesterolformulation at the lower (0.625 mg/mL) DNA concentration providing themost human HLA positive cells. Bone marrow cells showed high levels ofhuman HLA positive cells with all formulations tested.

In view of these results, a series of experiments were performed todemonstrate formulation-dependent targeting of DNA:lipid complexes tospleen and lung. Two formulations were used:

a. DOTIM:Cholesterol (1:1) DNA:Lipid ratio of 1:6 DNA concentration of0.625 mg/mL

b. DOTIM:DOPE (1:1) DNA:Lipid ratio of 1:1 DNA concentration of 1.5mg/mL

Each formulation was prepared as described in Examples 1 and 3 above,using a CAT-encoding construct, and were administered by intravenousinjection into the tail vein of cohorts of 3 ICR mice per testedformulation. Liposomes that were not complexed with DNA were injectedinto a separate cohort of 3 mice as a control.

Animals were sacrificed 1-2 days after injection and analyzed by CATassay of lung and spleen tissues as described above. The results ofthese experiments are shown in FIG. 12. CAT activity is expressed as thepercentage of total ¹⁴ C-chloramphenicol counts converted to acetylatedand diacylated forms associated with each tissue. As can be seen fromthe Figure, the DOTIM:Cholesterol formulation administered intravenouslyresulted in 96% (of over 1 million counts) being localized to lungtissue; 2% of the counts resulting from this formulation were found inthe spleen, and the rest were found in other tissues. In contrast, theDOTIM:DOPE formulation administered intravenously resulted in 91% (of160,000 counts) being localized to spleen tissue, with about 3% of thecounts being found in the lung and the rest being found in othertissues. These results demonstrate that this DOTIM:Cholesterolformulation specifically targets the DNA:lipid complex to the lung,while the DOTIM:DOPE formulation specifically targets DNA:lipidcomplexes to the spleen. In addition, these results show that CATactivity is about 10-fold more robust when delivered inDOTIM:Cholesterol complexes to the lung that the CAT activity resultingfrom DOTIM:DOPE complex-mediated delivery to spleen.

D. Intraperitoneal Delivery Formulations

Liposome formulations were developed for targeted gene delivery byintraperitoneal administration. DOTIM:Cholesterol formulations (1:1)were tested using a CAT-encoding construct at a DNA:lipid ratio of 1:1and a total DNA concentration in the complex of 2.5 mg/mL. 1 mL of theseDNA complexes were injected into the peritoneal cavity of each of 4mice; an equal volume of the liposome formulation not complexed withCAT-encoding DNA was injected into 4 mice in a separate cohort as acontrol. Peritoneal macrophages were isolated 24-48 h after injectionand tested for CAT activity as described above.

The results of these experiments are shown in FIG. 13. Peritonealmacrophages from control (untreated) mice showed essentially no CATactivity in this assay. Macrophages from mice administered the DNA:lipidcomplexes in this formulation intraperitoneally showed high levels ofCAT activity, demonstrating specific in vivo delivery of a functionalCAT gene using this formulation.

Spleens from these animals were also tested and CAT activity compared toperitoneal macrophages. These results are shown in FIG. 14, where it canbe seen that CAT activity in macrophages was much higher than in spleen,demonstrating specificity in targeting to these cells.

Pancreatic tissues were targeted for gene delivery using the DNA:lipidformulations of the invention as follows. A formulation comprising aCAT-expressing plasmid and DOTIM:DOPE (1:1), at a DNA:lipid ratio of1:1, and a total DNA concentration in the complex of 1.5 mg/mL wasinjected intraperitoneally into a cohort of 3 mice. Two mice wereinjected with the liposome formulation not complexed with DNA as acontrol. Pancreas and lung tissues were analyzed 24-48 hpost-administration for CAT activity as described above.

The results of these experiments are shown in FIG. 15. These resultsdemonstrate that this formulation specifically targets delivery ofCAT-encoding DNA constructs to the pancreas when administeredintraperitoneally.

A CAT-encoding recombinant construct was targeted to spleen using yetanother DNA:lipid formulation. Plasmid DNA was complexed withDOTIM:Cholesterol (1:1), at a DNA:lipid ratio of 1:1, and a total DNAconcentration in the complex of 2.5 mg/mL was injected intraperitoneallyinto a cohort of 3 mice. A separate cohort of mice were injected withthe liposome formulation not complexed with DNA as a control. Spleentissues from mice in each cohort were analyzed 24-48 hpost-administration for CAT activity as described above.

The results of these experiments are shown in FIG. 16. These resultsdemonstrate that this formulation specifically targets delivery ofCAT-encoding DNA constructs to spleen in vivo when administeredintraperitoneally.

The tissue specificity of intraperitoneal delivery was demonstrated bycomparison of two different formulations administered intraperitoneally.The following formulations were tested:

a. DOTIM:Cholesterol (1:1) DNA:Lipid ratio of 1:6 DNA concentration of0.625 mg/mL

b. DOTIM:DOPE (1:1) DNA:Lipid ratio of 1:1 DNA concentration of 1.5mg/mL

Each formulation was prepared as described in Examples 1 and 3 above,using a CAT-encoding construct, and were administered by intraperitonealinjection into the tail vein of cohorts of 3 ICR mice per testedformulation. Liposomes that were not complexed with DNA were injectedinto a separate cohort of 3 mice as a control.

Animals were sacrificed 1-2 days after injection and analyzed by CATassay of pancreas and spleen tissues as described above. The results ofthese experiments are shown in FIG. 17. CAT activity is expressed as thepercentage of total ¹⁴ C-chloramphenicol counts converted to acetylatedand diacylated forms associated with each tissue. As can be seen fromthe Figure, the DOTIM:DOPE formulation administered intraperitoneallyresulted in 96% (of 18 million counts) being localized to pancreatictissue; 3% of the counts resulting from this formulation were found inthe spleen, and the rest were found in other tissues. In contrast, theDOTIM:Cholesterol formulation administered intraperitoneally resulted in58% (of 28 million counts) being localized to spleen tissue, with about42% of the counts being found in the pancreas; essentially no CATactivity was observed in other tissues. These results demonstrate thatthis DOTIM:DOPE formulation specifically targets the DNA:lipid complexto the pancreas when administered intraperitoneally, while theDOTIM:Cholesterol formulation specifically targets DNA:lipid complexesto the pancreas and spleen.

E. Direct Delivery Formulations

Liposome formulations were developed for targeted gene delivery bydirect injection into tissues. DOTIM:Cholesterol formulations (1:1) weretested using a CAT-encoding construct at a DNA:lipid ratio of 1:1 and atotal DNA concentration in the complex of 2.5 mg/mL. This formulationwas directly injected in 1.5 mL into a human prostate ex corpora, andthe assayed by CAT assay as described above.

The results of this experiment are shown in FIG. 18. These resultsdemonstrate that gene delivery can be mediated by direct injection ofDNA:lipid complexes on the invention into human tissues.

F. Comparison of Intravenous and Intraperitoneal Administration Routes

The effect of administration route on targeted delivery of CAT-encodingplasmid DNA using a single DNA:lipid complex formulation was determined.DOTIM:Cholesterol formulations (1:1) were tested using a CAT-encodingconstruct at a DNA: lipid ratio of 1:1 and a total DNA concentration inthe complex of 2.5 mg/mL. Cohorts of 3 mice were either injectedintravenously in the tail vein, or intraperitoneally. Spleen and lungtissues were analyzed 24-48 h post-administration for CAT activity asdescribed above. The results of these experiments are shown in FIG. 19.It can be seen from the Figure that the highest CAT activity levels wereachieved in lung tissue following intravenous administration of theformulation. However, CAT activity after intraperitoneal administrationwas relatively higher in spleen than in lung. These results demonstratethat tissue-specific targeting of DNA delivery can be achieved with thesame efficacious formulation of DNA:lipid complexes, and that thetargeted site can be influenced by the route of administration.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

We claim:
 1. A pharmaceutical composition comprising a formulation of acomplex of a recombinant expression construct and a mixture of a neutrallipid and1-(2-(oleoyloxy)ethyl)-2-oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazoliniumchloride as a cationic lipid in a pharmaceutically acceptable carriersuitable for administration to an animal by intravenous, intraperitonealor direct injection into a tissue in the animal, wherein:(a) therecombinant expression construct comprises a nucleic acid encoding aprotein and wherein said nucleic acid is operatively linked to geneexpression regulatory elements whereby the protein encoded by thenucleic acid is expressed in cells in a tissue in an animal; and (b) thecationic lipid and the neutral lipid are present in the complex at amolar ratio of about 1:1, the DNA and the cationic lipid are present inthe complex in a ratio of from about 1:1 to about 1:8 μg DNA/nmolecationic lipid, and the nucleic acid comprising the recombinantexpression construct is present in the complex at a concentration ofabout 0.5 mg/mL to about 5 mg/mL.
 2. The pharmaceutical composition ofclaim 1 wherein the neutral lipid is cholesterol ordioleoylphosphatidylethanolamine.
 3. The pharmaceutical composition ofclaim 1 wherein the complex of the recombinant expression construct anda mixture of a neutral lipid and a cationic lipid comprises a ratio ofDNA to cationic lipid of about 1:1 μg DNA/nmole cationic lipid.
 4. Amethod of introducing a recombinant expression construct into a cellwithin a lung in an animal, the method comprising the step ofadministering the pharmaceutical composition of claim 1 to the animal byintravenous injection, wherein the neutral lipid is cholesterol, thecationic lipid and the neutral lipid are present in a molar ratio ofabout 1:1, the complex of the recombinant expression construct and amixture of a neutral lipid and a cationic lipid comprises a ratio of DNAto cationic lipid of about 1:6 μg DNA/nmole cationic lipid, and the DNAconcentration in the DNA:lipid complexes is from about 0.5 mg/mL toabout 1 mg/mL.
 5. A method of introducing a recombinant expressionconstruct into a cell within a spleen in an animal, the methodcomprising the step of administering the pharmaceutical composition ofclaim 1 to the animal by intravenous injection, wherein the neutrallipid is dioleoylphosphatidyl-ethanolamine, the cationic lipid and theneutral lipid are present in a molar ratio of about 1:1, the complex ofthe recombinant expression construct and a mixture of a neutral lipidand a cationic lipid comprises a ratio of DNA to cationic lipid of about1:1 μg DNA/nmole cationic lipid, and the DNA concentration in theDNA:lipid complexes is from about 1 mg/mL to about 2.5 mg/mL.
 6. Amethod of introducing a recombinant expression construct into a cellthat is a peritoneal macrophage in an animal, the method comprising thestep of administering the pharmaceutical composition of claim 1 to theanimal by intraperitoneal injection, wherein the neutral lipid ischolesterol, the cationic lipid and the neutral lipid are present in amolar ratio of about 1:1, the complex of the recombinant expressionconstruct and a mixture of a neutral lipid and a cationic lipidcomprises a ratio of DNA to cationic lipid of about 1:1 μg DNA/nmolecationic lipid, and the DNA concentration in the DNA:lipid complexes isfrom about 1 mg/mL to about 2.5 mg/mL.
 7. A method of introducing arecombinant expression construct into a cell within a spleen in ananimal, the method comprising the step of administering thepharmaceutical composition of claim 1 to the animal by intraperitonealinjection, wherein the neutral lipid is cholesterol, the cationic lipidand the neutral lipid are present in a molar ratio of about 1:1, thecomplex of the recombinant expression construct and a mixture of aneutral lipid and a cationic lipid comprises a ratio of DNA to cationiclipid of about 1:1 μg DNA/nmole cationic lipid, and the DNAconcentration in the DNA-lipid complexes is from about 1 mg/mL to about2.5 mg/mL.
 8. A method of introducing a recombinant expression constructinto a cell within a pancreas in an animal, the method comprising thestep of administering the pharmaceutical composition of claim 2 to theanimal by intraperitoneal injection, wherein the neutral lipid isdioleoylphosphatidylethanolamine, the cationic lipid and the neutrallipid are present in a molar ratio of about 1:1 the complex of therecombinant expression construct and a mixture of a neutral lipid and acationic lipid comprises a ratio of DNA to cationic lipid of about 1:1μg DNA/nmole cationic lipid, and the DNA concentration in the DNA:lipidcomplexes is from about 1 mg/mL to about 2.5 mg/mL.
 9. A method ofintroducing a recombinant expression construct into a cell within atissue in an animal, the method comprising the step of administering thepharmaceutical composition of claim 1 to the animal by direct injectioninto the tissue in the animal, wherein the neutral lipid is cholesterol,the cationic lipid and the neutral lipid are present in a molar ratio ofabout 1:1 the complex of the recombinant expression construct and amixture of a neutral lipid and a cationic lipid comprises a ratio of DNAto cationic lipid of about 1:1 μg DNA/nmole cationic lipid, and the DNAconcentration in the DNA:lipid complexes is from about 1 mg/ml to about2.5 mg/mL.